Killing of Leishmania donovani by activated liver macrophages from resistant and susceptible strains of mice

Leishmania donovani is an obligate intracellular protozoan parasite of macrophages; liver macrophages are, however, the only population of cells which express the resistant Lsh gene phenotype when these cells are infected in vitro. It was of interest to study in vitro the action of Con A-stimulated spleen cell lymphokines (LK) to protect or to cure liver macrophages from infection by L. donovani. Liver and peritoneal macrophages (PEC) from resistant (C57L/J) and susceptible (C57BL/6J) mice were infected in vitro with promastigotes before or after LK treatment; the percentage of infected macrophages was determined 4, 24, 48 and 72 h post-infection. Both macrophage populations were protected or cured by treatment with lymphokines; the cells of the resistant strain were protected or cured more effectively than those of the susceptible strain. The capacity for cure or for protection following LK treatment of liver and PEC macrophages was similar within each strain. Supernatants from the IL-2-produced MLA-144 cell line had no effect to protect or cure macrophages. This study indicates that the response of macrophages to the action of LK is also important in determining the susceptibility of mice to L. donovani; this model in vitro provides a good approximation of the response of macrophages to therapy.     

Trypanosoma cruzi: regulation of mitogenic responses during infection in genetically resistant and susceptible inbred mouse strains

The outcome of Trypanosoma cruzi infection in inbred strains of mice is under genetic control. The lymphocyte responses to T-cell mitogens and their regulation were investigated in strains of mice resistant or susceptible to T. cruzi. Six to eight days after the inoculation of T. cruzi, resistant and susceptible mice had depressed responses to T-cell mitogens. In resistant B6 mice, suppression was maximal 18 days after infection and it persisted for at least 320 days. The duration of immunosuppression correlated with the persistence of a subpatent parasitemia. In cell mixing experiments, it was determined that the concanavalin A (Con A) responses in the resistant B6 and B6C3F1 mouse strains were suppressed by highly active T-suppressor cells. In the susceptible C3H mice, intense suppression of the Con A responses was detected 14 days after inoculation of T. cruzi. Nevertheless, only weak suppressor cell activity was detected in the infected C3H mice, and suppression was not abrogated by passage through a nylon wool column nor by treatment with antitheta antibodies and complement. Thus, it was suggested that, during the course of infection with T. cruzi, splenic T cells from C3H mice acquired a block in the metabolic pathway for cellular activation by Con A. The influences of T. cruzi epimastigotes on the Con A responses of spleen cells from uninfected mice were then studied. The Con A responses of spleen cells from C3H mice were depressed in the presence of epimastigotes, whereas they were either unaffected or enhanced in spleen cells from B6 mice. Hence, the immunoregulatory events provoked by T. cruzi infection differed in genetically resistant and susceptible mice, and lymphocytes from C3H mice were predisposed to a parasite-induced block in the responses to Con A. Thus, the gene(s) determining the outcome of infection with T. cruzi may be phenotypically expressed through an influence on immunoregulatory events.     

Comparison of lung virus titers in susceptible and resistant inbred mouse strains against Sendai virus infection

Susceptibility of inbred mouse strains to Sendai virus (Mol strain) infection was studied. Although some mouse strains showed age differences in susceptibility between 3-to 4-week-old and 7-to 8-week-old mice, such age differences in susceptibility were not observed in susceptible DBA/2N and resistant BALB/cA mice. In 7-to 8-week-old mice, remarkable strain differences were observed in mortality and intensity of the lung lesions, but not in lung virus titers and serum antibody, between resistant BALB/cA and susceptible DBA/2N mice.     

Pleiotropic effects of the Bcg gene. I. Antigen presentation in genetically susceptible and resistant congenic mouse strains

The Ag-presentation ability of Bcgr and Bcgs spleen cells was studied in two sets of Bcg-congenic systems; namely, the BALB/c-BALB/c.Bcgr pair and the B10.A-B10.A-Bcgr pair, by using three sonicated soluble bacterial Ag (mycobacterium bovis bacillus Calmette-Guérin, Salmonella typhimurium, and Brucella abortus) as well as a particulate Ag (heat-killed Escherichia coli). Pulsed Bcgr spleen cells were shown to induce a stronger proliferation of the T cell-indicator system than their Bcgs counterparts. No difference in Ag-presenting ability could be shown between Bcgr and Bcgs peritoneal macrophages from normal animals. However, elicited peritoneal macrophages from immune Bcgr mice were superior in their Ag-presentation ability. Differences at the level of Ag presentation of Bcgr and Bcgs splenic cells were investigated further. Depletion of T cells and B cells did not alter the differences in Ag-presenting ability between Bcgr and Bcgs spleen cells. Furthermore, splenic dendritic cells of Bcgr or Bcgs allelic types were equally efficient in presenting bacillus Calmette-Guérin Ag to accessory cell-depleted T cells. In a final experiment, it was shown that spleen macrophages were the cell type involved in the superior Ag presentation by Bcgr splenic cells.     

Plasmodium yoelii: antibody response in resistant and susceptible mouse strains

The numbers of antigen-reactive antibody-secreting cells, levels of parasite antigen-specific serum antibodies and numbers of red blood cells staining positive for surface immunoglobulin were determined for susceptible and resistant mouse strains following infection with Plasmodium yoelii 17x. As a control, these parameters also were measured using antigen prepared from normal red blood cells. The relatively susceptible C57BL/6 mice produced more antigen-specific antibody-secreting cells and had higher levels of immunoglobulin positive red blood cells than did DBA/2 mice, but the DBA/2 mice had more antigen-specific IgG in their sera. Both mouse strains possessed cells secreting antibody reactive with soluble normal red blood cell antigen; however, C57BL/6 mice had more IgG positive unparasitized RBC than did DBA/2 mice. Despite possessing fewer antibody positive normal RBC, DBA/2 mice had significantly higher levels of serum antibodies that reacted with soluble red blood cell antigen. These data indicate that levels of serum antibody may not reflect the amounts of antibody produced and that use of any single assay to assess the magnitude of the antibody response may give rise to misleading results.     

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.     

Congenic strains of mice susceptible and resistant to mouse hepatitis virus.

A congenic strain of C3HSS mice, which is histocompatible with C3H mice but differs from them in susceptibility to mouse hepatitis virus (MHV), has been developed by introducing the gene for susceptibility to the MHV-PRI virus from the PRI mice. This was accomplished by continual back-crossing of the hybrids to the C3H mice, but at the same time by selection of susceptibility by use of macrophage culture tests. After 20 back-crosses, a strain homozygous for susceptibility was produced by brother-sister mating of individual mice whose potential for carrying the recessive gene for resistance was tested in progeny. Since the original choice of mice for breeding was based on in vitro macrophage susceptibility, and since highly susceptible mice were developed on the same basis, it seems evident that macrophage susceptibility is an integral aspect of mouse susceptibility. The continued production of almost 50% susceptible mice in the back-crosses is further evidence of the dominant one-locus explanation of genetic susceptibility to this agent. Incomplete penetrance may also be present in 8 and 9 week old mice of the C3HSS strain since there was a sharp decrease in susceptibility of these mice even though their macrophages in culture maintained full susceptibility.     

Gene expression changes in the host response between resistant and susceptible inbred mouse strains after influenza A infection

Inbred mouse strains exhibit differences in susceptibility to influenza A infections. However, the molecular mechanisms underlying these differences are unknown. Therefore, we infected a highly susceptible mouse strain (DBA/2J) and a resistant strain (C57BL/6J) with influenza A H1N1 (PR8) and performed genome-wide expression analysis. We found genes expressed in lung epithelium that were specifically down-regulated in DBA/2J mice, whereas a cluster of genes on chromosome 3 was only down-regulated in C57BL/6J. In both mouse strains, chemokines, cytokines and interferon-response genes were up-regulated, indicating that the main innate immune defense pathways were activated. However, many immune response genes were up-regulated in DBA/2J much stronger than in C57BL/6J, and several immune response genes were exclusively regulated in DBA/2J. Thus, susceptible DBA/2J mice showed a hyper-inflammatory response. This response is similar to infections with highly pathogenic influenza virus and may serve as a paradigm for a hyper-inflammatory host response to influenza A virus.     

Importance of lymphokines in the control of multiplication and dispersion of Leishmania donovani within liver macrophages of resistant and susceptible mice

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. The immunosuppressant cyclosporin A (CsA), which inhibits the production of interleukin (IL)-1, IL-2, and interferon-gamma, increased infections 3-fold without affecting expression of the Lsh gene. The objective of this study was to determine how activation of macrophages by lymphokines affects the multiplication and propagation of the parasite within liver macrophages. Susceptible C57BL/6J and resistant C57L/J mice were treated with 200 mg/kg CsA and then infected intravenously with 10(7) amastigotes. Two weeks later macrophages were collected from the liver by perfusion, plated on coverslips, and incubated for 4, 24, and 48 hr. The percentage of infected macrophages and the number of amastigotes/100 cells were determined after staining the cells with Giemsa's stain. The number of infected macrophages and amastigotes per macrophage was significantly greater in animals of both strains that had been treated with CsA. This study demonstrated clearly that lymphokines or other soluble mediators produced by T cells act, in part, to control infection by L. donovani by minimizing both multiplication within macrophages and their dispersion.     

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.     

Theiler's virus induces the MIP-2 chemokine (CXCL2) in astrocytes from genetically susceptible but not from resistant mouse strains

The murine encephalomyelitis virus of Theiler (TMEV) induces demyelination in susceptible strains of mice by a CD4(+) Th1 T cell mediated immunopathologic process. We focused on the production of one chemokine, the macrophage inflammatory protein-2 (MIP-2 or CXCL2), by cultured mouse astrocytes infected with the BeAn strain of TMEV. Analysis of a murine genome DNA hybridized with cRNA from mock- and TMEV-infected astrocytes, revealed up-regulation of three sequences encoding MIP-2. Northern blot analysis indicated increased MIP-2 mRNA expression. Levels of MIP-2 in the supernatants of infected cells as detected by ELISA, varied directly with the multiplicity of infection used. This secreted CXCL2 was biologically active inducing chemoattraction of neutrophils but not of lymphocytes. CXCL2 was specifically induced by TMEV infection, since induction was inhibited by anti TMEV antibodies. The inflammatory cytokines, IL-1alpha and TNF-alpha, which are also induced in astrocytes by TMEV, were very potent inducers of CXCL2. Nevertheless, both mechanisms of induction follows different pathways as antibodies to both cytokines fails to inhibit TMEV-induced CXCL2 up-regulation. Sera from TMEV-infected SJL/J mice with chronic demyelination, but not from BALB/c TMEV-resistant mice, revealed CXCL2 at the peak of clinical disease. Our main novel finding is the strain-dependent differences in CXCL2 expression both in vitro and in vivo. This suggest an role for this chemokine in attracting immune cells within the CNS, which in turn, might trigger demyelination in this experimental model of MS.     

Studies on stibanate resistant Leishmania donovani isolates of Indian origin

Studies with 26 clones of L. donovani promastigotes derived from three different Indian isolates indicated that wild type parasites are mixture of stibanate sensitive and resistant cells. Both forms of the parasite were resistant to the drug. Infection with resistant parasites appears to be the primary reason of high rate of pentavalent antimony unresponsiveness among Indian kala-azar patients. It was observed that the resistant parasites originated as a result of irregular and often incomplete treatment of kala-azar patients with pentavalent antimonials.     

IL-12Rbeta2-deficient mice of a genetically resistant background are susceptible to Leishmania major infection and develop a parasite-specific Th2 immune response

The cytokine IL-12 plays a critical role in inducing the production of IFN-gamma from T and NK cells and in the polarization of T cells towards the Th1 phenotype. IL-12 is comprised of two subunits (IL-12p40 and IL-12p35) that together form the biologically active p70 molecule, and IL-12 functions via binding to a heterodimeric receptor (IL-12Rbeta1 and IL-12Rbeta2). Previous studies utilizing mice deficient for either the IL-12 cytokine or the IL-12-induced signaling molecule STAT4 have established a critical role for IL-12 during infection with Leishmania major. However, these studies warrant careful re-interpretation in light of the recent discovery of the IL-12-related cytokine, IL-23, which utilizes the IL-12p40 chain in combination with an IL-12p35-related molecule, called p19, and a receptor comprised of the IL-12Rbeta1 chain plus a unique chain referred to as IL-23R. We analyzed the course of L. major infection in mice deficient for the IL-12-specific IL-12Rbeta2 subunit in order to assess the role of IL-12 signaling without disruption of the IL-23 pathway. After infection with L. major, IL-12Rbeta2KO mice of a resistant background (C57Bl/6) developed large cutaneous lesions similar to those developed by susceptible BALB/c mice. Draining lymph node cells from L. major-infected IL-12Rbeta2KO mice released the Th2 cytokines IL-4 and IL-5 after in vitro stimulation with Leishmania lysate but were completely devoid of IFN-gamma, consistent with a default towards a strong parasite-specific Th2 response. L. major-infected IL-12Rbeta2KO mice were also devoid of parasite-specific IgG2a antibodies, and interestingly, their footpad lesions ulcerated earlier than those of susceptible BALB/c mice.     

Leishmania major: analysis of lymphocyte and macrophage cellular phenotypes during infection of susceptible and resistant mice

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.     

B cell-deficient mice are highly resistant to Leishmania donovani infection, but develop neutrophil-mediated tissue pathology

Resolution of Leishmania infection is T cell-dependent, and B lymphocytes have been considered to play a minimal role in host defense. In this study, the contribution of B lymphocytes to the response against Leishmania donovani was investigated using genetically modified IgM transmembrane domain (muMT) mutant mice, which lack mature B lymphocytes. When compared with wild-type mice, muMT mice cleared parasites more rapidly from the liver, and infection failed to establish in the spleen. The rapid clearance of parasites in muMT mice was associated with accelerated and more extensive hepatic granuloma formation compared with wild-type mice. However, the liver of infected muMT mice also showed signs of destructive pathology, associated with the presence of increased numbers of neutrophils. The role of neutrophils in controlling parasite growth in the viscera was determined by depletion with the mAb RB6-8C5. This treatment led to a dramatic enhancement of parasite growth in both the liver and spleen of muMT and wild-type mice. As assessed by transfer of both normal and chronic-infection serum, Ig protects microMT mice from destructive hepatic pathology, but minimally alters their resistance compared with wild-type mice. However, adoptive transfer of CD4+ and CD8+ T cells into recombinase activating gene 1 (RAG1-/-) recipients, suggested that T cell function was not altered by maturation in a B cell-deficient environment. Taken together, these data suggest an inhibitory role for B lymphocytes in resistance to L. donovani unrelated to the presence or absence of Ig. However, Ig protects muMT mice from the exaggerated pathology that occurs during infection.     

Differential interleukin-6 mRNA expression in Nippostrongylus brasiliensis infection of susceptible and resistant strains of mice

Intestinal parasitic infection is still a major problem in humans and animals, yet host immunity against gut parasitic infection remains partially understood. Eosinophilia and mastocytosis are features of such infection that have been shown to be genetically controlled. The expression of IL-6 is detected in eosinophils, mast cells and neutrophils and may be responsible for the regulation of leucocytes at infective sites. The relationships between IL-6 expression, eosinophilia, mastocytosis and host immunity remain unclear. In the present report, a close correlation between IL-6 mRNA+ cells, eosinophilia, mastocytosis and worm expulsion is demonstrated, which may indicate a role for IL-6 in regulation of host immunity against intestinal parasite infection.